Parkinson?s disease (PD) is a progressive neurodegenerative disease that results in severe movement disorders and gastrointestinal symptoms such as gastroparesis and constipation. The cause of PD is unknown and there is no cure for the disease. PD is common in the VA and over 40,000 veterans are treated in VA medical facilities each year. There is evidence that environmental toxins such as the pesticide, rotenone, and the herbicide, Agent Orange, may play a role in causing Parkinson?s disease. Military veterans exposed to Agent Orange have an increased incidence of PD and the Institute of Medicine concluded in its report ?Veterans and Agent Orange: Update 2008?, that exposure to Agent Orange and other herbicides used during the Vietnam War is associated with an increased chance of developing Parkinson's disease. As a result, VA recognized that PD was associated with exposure to Agent Orange during military service. Despite the association between environmental toxin exposure and PD, how environmental toxins cause PD is unknown. The pathological hallmarks of PD are cytoplasmic inclusions known as Lewy bodies in the brain and enteric nervous system. These inclusions are associated with degeneration of dopaminergic neurons in the substantia nigra pars compacta which produces the distinctive disorders of movement and vagal nerve dysfunction. The major component of Lewy pathology is aggregated ?-synuclein, a synaptic protein with the propensity to misfold and aggregate. Misfolded ?-synuclein plays a critical role in PD pathogenesis and recent evidence supports a model in which propagation of Lewy pathology occurs via cell-to-cell transmission of misfolded ?-synuclein onto recipient cells. Misfolded ?-synuclein recruits native ?-synuclein in the recipient cell and acts as a template or nidus for the development of aggregates that eventually lead to formation of Lewy bodies and ultimately PD. There is evidence that PD starts in the gut before affecting the brain. For example, ?-synuclein, which is found in an abnormal form in the brains of PD patients, appears in abnormal form in gut nerves before it appears in the brain. Moreover, cutting the vagus nerve (vagotomy) reduces the risk of developing PD. Nevertheless, understanding how environmental toxins cause the abnormal form of ?- synuclein to form in the nervous system of the gut is lacking. Enteroendocrine cells (EECs) are specialized sensory cells in the lining of the gut. In this location, EECs are exposed to food and ingested environmental toxins. We recently made two important discoveries. First, we discovered that EECs connect to nerves, thus providing a direct route from the intestine to the brain. Second, we discovered that EECs express ?-synuclein and may be the source of abnormal ?-synuclein that could spread to the brain. To investigate this possibility, we will perform proof-of-concept studies to characterize ?-synuclein in EECs using a model of EECs in vitro, intestinal organoids that contain EECs, and in mouse models in which animals are exposed to the environmental toxins rotenone and Agent Orange. We have developed tools to localize and characterize different forms of ?-synuclein in EECs of the gut and in nerves. We will determine if potential neurotoxins affect ?-synuclein gene expression, protein abundance, and aggregation in an EEC cell line and in intestinal organoids in culture. We will then perform detailed feeding studies to determine if rotenone and Agent Orange cause similar changes to ?-synuclein in their EECs. Therefore, these studies will test the hypothesis that environmental toxins induce abnormal ?-synuclein formation in EECs that then spread to the nervous system to cause PD. This new knowledge will form the basis for future studies to characterize ?-synuclein in EECs of patients with PD. Ultimately these studies could lead to the development of a diagnostic test for the very earliest stages of Parkinson?s disease well before the disease can be diagnosed currently. In addition, if our theories are supported, this could lead to new methods of prevention or treatment for Parkinson?s disease.